LampPort: a handheld digital microfluidic device for loop-mediated isothermal amplification (LAMP).

A major goal in the development of point-of-care (POC) devices is to build them as portable to provide a rapid and effective determination for disease pathogens. In nucleic acid testing, an optical detection system used to monitor the product of nucleic acid amplification has always been a bulky accessory. In this work, we developed a handheld, automatic and detection system-free thermal digital microfluidic (DMF) device for DNA detection by loop-mediated isothermal amplification (LAMP). Droplet manipulation and real-time temperature control systems were integrated into a handheld device. The control software could be installed into any tablet and communicate with the device via Bluetooth. In the experimentation, we loaded 2-µl samples with an electrowetting force into sandwich-structured DMF chips, thereby considerably reducing reagent consumptions. After an on-chip LAMP reaction, we added a highly concentrated SYBR Green I droplet and mixed it with a reaction droplet to enable product detection with the naked eye. This step prevented aerosol contamination by avoiding the exposure of the reaction droplet to the air. Using a blood parasite Trypanosoma brucei as a model system, this system showed similar results as a commercial thermal cycler and could detect 40 copies per reaction of the DNA target. This low-cost, compact device removed the bulky optical system for DNA detection, thus enabling it to be well suited for POC testing.

Characterization of in vitro metabolites of irisflorentin by rat liver microsomes using high-performance liquid chromatography coupled with tandem mass spectrometry.

Belamcanda chinensis has been extensively used as antibechic, expectorant and anti-inflammatory agent in traditional medicine. Irisflorentin is one of the major active ingredients. However, little is known about the metabolism of irisflorentin so far. In this work, rat liver microsomes (RLMs) were used to investigate the metabolism of this compound for the first time. Seven metabolites were detected. Five of them were identified as 6,7-dihydroxy-5,3',4',5'-tetramethoxy isoflavone (M1), irigenin (M2), 5,7,4'-trihydroxy-6,3',5'-trimethoxy isoflavone (M3), 6,7,4'-trihydroxy-5,3',5'-trimethoxy isoflavone (M4) and 6,7,5'-trihydroxy-5,3',4'-trimethoxy isoflavone (M5) by means of NMR and/or HPLC-ESI-MS. The structures of M6 and M7 were not elucidated because they produced no MS signals. The predominant metabolite M1 was noted to be a new compound. Interestingly, it was found to possess anticancer activity much higher than the parent compound. The enzymatic kinetic parameters of M1 revealed a sigmoidal profile, with Vmax = 12.02 µm/mg protein/min, Km = 37.24 µm, CLint = 0.32 µL/mg protein/min and h = 1.48, indicating the positive cooperation. For the first time in this work, a new metabolite of irisflorentin was found to demonstrate a much higher biological activity than its parent compound, suggesting a new avenue for the development of drugs from B. chinensis, which was also applicable for other herbal plants. Copyright © 2016 John Wiley & Sons, Ltd.

Quick and selective extraction of Z-ligustilide from Angelica sinensis using magnetic multiwalled carbon nanotubes.

A facile and highly efficient magnetic solid-phase extraction method has been developed for Z-ligustilide, the major therapeutic agent in Angelica sinensis. The solid-phase adsorbent material used was prepared by conjugating carbon nanotubes with magnetic Fe3 O4 nanoparticles via a hydrothermal reaction. The magnetic material showed a high affinity toward Z-ligustilide due to the π-π stacking interaction between the carbon nanotubes and Z-ligustilide, allowing a quick and selective exaction of Z-ligustilide from complex sample matrices. Factors influencing the magnetic solid-phase extraction such as the amount of the added adsorbent, adsorption and desorption time, and desorption solvent, were investigated. Due to its high extraction efficiency, this method was proved highly useful for sample cleanup/enrichment in quantitative high-performance liquid chromatography analysis. The proposed method had a linear calibration curve (R(2) = 0.9983) over the concentration between 4 ng/mL and 200 µg/mL Z-ligustilide. The accuracy of the method was determined by the recovery, which was from 92.07 to 104.02%, with the relative standard deviations >4.51%.

Metabolic study of Angelica dahurica extracts using a reusable liver microsomal nanobioreactor by liquid chromatography-mass spectrometry.

Highly active and recoverable nanobioreactors prepared by immobilizing rat liver microsomes on magnetic nanoparticles (LMMNPs) were utilized in metabolic study of Angelica dahurica extracts. Five metabolites were detected in the incubation solution of the extracts and LMMNPs, which were identified by means of HPLC-MS as trans-imperatorin hydroxylate (M1), cis-imperatorin hydroxylate (M2), imperatorin epoxide (M3), trans-isoimperatorin hydroxylate (M1') and cis-isoimperatorin hydroxylate (speculated M2'). Compared with the metabolisms of imperatorin and isoimperatorin, it was found that the five metabolites were all transformed from these two major compounds present in the plant. Since no study on isoimperatorin metabolism by liver microsomal enzyme system has been reported so far, its metabolites (M1' and M3') were isolated by preparative HPLC for structure elucidation by (1) H-NMR and MS(2) analysis. M3' was identified as isoimperatorin epoxide, which is a new compound as far as its chemical structure is concerned. However, interestingly, M3' was not detected in the metabolism of the whole plant extract. In addition, a study with known chemical inhibitors on individual isozymes of the microsomal enzyme family revealed that CYP1A2 is involved in metabolisms of both isoimperatorin and imperatorin, and CYP3A4 only in that of isoimperatorin.

A sandwich FRET biosensor for lysozyme detection based on peptide-functionalized gold nanoparticles and FAM-labeled aptamer.

Lysozyme (LYZ) plays a crucial role in the body's immune defense system. Monitoring LYZ levels can provide valuable insights into the diagnosis and severity assessment of various diseases. Traditionally, antibody-based sandwich assays are employed for LYZ detection, but they are often time-consuming and operationally complicated. In this research, a novel sandwich FRET biosensor was developed, which enables rapid detection of LYZ based on peptide-functionalized gold nanoparticles (pAuNPs) and FAM-labeled aptamer (Apt-FAM). Initially, a mixture of Apt-FAM and pAuNPs resulted in partial quenching of the Apt-FAM fluorescence emission through an inner filter effect (IFE), with negligible energy transfer because of the electrostatic repulsion between the negatively charged pAuNPs and Apt-FAM. The introduction of LYZ into the mixture drove the specific binding of Apt-FAM and pAuNPs to LYZ, facilitating the formation of a pAuNPs-LYZ-aptamer sandwich structure. The formation of this complex drew the pAuNPs and Apt-FAM into close enough proximity to enable FRET to occur, which in turn effectively quenched the fluorescence emission of FAM. The decrease in FAM fluorescence intensity was correlated with the increasing concentration of LYZ. Thus, a sandwich FRET biosensor was successfully developed for LYZ detection with a linear detection range of 0-1.75 µM and a detection limit of 85 nM. Additionally, the biosensor allowed visual detection of LYZ in a 96-well microplate, with a rapid response time of just 15 s. This study introduces a innovative sandwich FRET biosensor that combines aptamer and peptide recognition elements, offering a fast and antibody-free method for protein detection.

Analysis and numerical investigation of bile flow dynamics within the strictured biliary duct.

The mechanics of bile flow in the biliary system plays an important role in studying bile stasis and gallstone formation. Bile duct stricture is an abnormal phenomenon that refers to the bile duct getting smaller or narrower. The main objective of this study is to study the influence of stricture on bile flow dynamics using numerical methods. We employed a numerical Computational Fluid Dynamics model of the bile flow within a strictured hepatic duct. We studied and compared the influence of stricture severity, stricture length, eccentricity, and bile flow property on the bile flow dynamics. The bile flow velocity, pressure distribution, pressure drop, and wall shear stress are provided in detail. The stricture alters the normal bile flow pattern and increases flow resistance. At the location upstream and downstream of the stricture, bile flow slows down. In the area of the stricture throat, bile flow is accelerated, and recirculation forms behind the stricture. The maximum pressure drop of the biliary system increases with the stricture length. The eccentricity makes the flow deflect away from the duct's centerline. The behavior of the deflected flow is significantly altered downstream of the stricture. Such bile flow behavior as deceleration and recirculation may lead to cholestasis. Stricture alters bile flow in the biliary tract, causing changes in biliary hydrodynamic indexes, which could potentially serve as an omen for gallstone formation and other related diseases. The consideration of the bile duct stricture could lead to better patient stratification.

Investigation on submicron particle separation and deflection using tilted-angle standing surface acoustic wave microfluidics.

With the development of in vitro diagnostics, extracting submicron scale particles from mixed body fluids samples is crucial. In recent years, microfluidic separation has attracted much attention due to its high efficiency, label-free, and inexpensive nature. Among the microfluidic-based separation, the separation based on ultrasonic standing waves has gradually become a powerful tool. A microfluid environment containing a tilted-angle ultrasonic standing surface acoustic wave (taSSAW) field has been widely adapted and designed to separate submicron particles for biochemical applications. This paper investigated submicron particle defection in microfluidics using taSSAWs analytically. Particles with 0.1-1 µm diameters were analyzed under acoustic pressure, flow rate, tilted angle, and SSAW frequency. According to different acoustic radiation forces acting on the particles, the motion of large-diameter particles was more likely to deflect to the direction of the nodal lines. Decreasing the input flow rate or increasing acoustic pressure and acoustic wave frequency can improve particle deflection. The tilted angle can be optimized by analyzing the simulation results. Based on the simulation analysis, we experimentally showed the separation of polystyrene microspheres (100 nm) from the mixed particles and exosomes (30-150 nm) from human plasma. This research results can provide a certain reference for the practical design of bioparticle separation utilizing acoustofluidic devices.

Review on bile dynamics and microfluidic-based component detection: Advancing the understanding of bilestone pathogenesis in the biliary tract.

Bilestones are solid masses found in the gallbladder or biliary tract, which block the normal bile flow and eventually result in severe life-threatening complications. Studies have shown that bilestone formation may be related to bile flow dynamics and the concentration level of bile components. The bile flow dynamics in the biliary tract play a critical role in disclosing the mechanism of bile stasis and transportation. The concentration of bile composition is closely associated with processes such as nucleation and crystallization. Recently, microfluidic-based biosensors have been favored for multiple advantages over traditional benchtop detection assays for their less sample consumption, portability, low cost, and high sensitivity for real-time detection. Here, we reviewed the developments in bile dynamics study and microfluidics-based bile component detection methods. These studies may provide valuable insights into the bilestone formation mechanisms and better treatment, alongside our opinions on the future development of in vitro lithotriptic drug screening of bilestones and bile characterization tests.

Bio-Inspired Microreactors Continuously Synthesize Glucose Precursor from CO2 with an Energy Conversion Efficiency 3.3 Times of Rice.

Excessive CO2 and food shortage are two grand challenges of human society. Directly converting CO2 into food materials can simultaneously alleviate both, like what green crops do in nature. Nevertheless, natural photosynthesis has a limited energy efficiency due to low activity and specificity of key enzyme D-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). To enhance the efficiency, many prior studies focused on engineering the enzymes, but this study chooses to learn from the nature to design more efficient reactors. This work is original in mimicking the stacked structure of thylakoids in chloroplasts to immobilize RuBisCO in a microreactor using the layer-by-layer strategy, obtaining the continuous conversion of CO2 into glucose precursor at 1.9 nmol min-1 with enhanced activity (1.5 times), stability (≈8 times), and reusability (96% after 10 reuses) relative to the free RuBisCO. The microreactors are further scaled out from one to six in parallel and achieve the production at 15.8 nmol min-1 with an energy conversion efficiency of 3.3 times of rice, showing better performance of this artificial synthesis than NPS in terms of energy conversion efficiency. The exploration of the potential of mass production would benefit both food supply and carbon neutralization.

Drug screening on digital microfluidics for cancer precision medicine.

Drug screening based on in-vitro primary tumor cell culture has demonstrated potential in personalized cancer diagnosis. However, the limited number of tumor cells, especially from patients with early stage cancer, has hindered the widespread application of this technique. Hence, we developed a digital microfluidic system for drug screening using primary tumor cells and established a working protocol for precision medicine. Smart control logic was developed to increase the throughput of the system and decrease its footprint to parallelly screen three drugs on a 4 × 4 cm2 chip in a device measuring 23 × 16 × 3.5 cm3. We validated this method in an MDA-MB-231 breast cancer xenograft mouse model and liver cancer specimens from patients, demonstrating tumor suppression in mice/patients treated with drugs that were screened to be effective on individual primary tumor cells. Mice treated with drugs screened on-chip as ineffective exhibited similar results to those in the control groups. The effective drug identified through on-chip screening demonstrated consistency with the absence of mutations in their related genes determined via exome sequencing of individual tumors, further validating this protocol. Therefore, this technique and system may promote advances in precision medicine for cancer treatment and, eventually, for any disease.

Digital Microfluidics with an On-Chip Drug Dispenser for Single or Combinational Drug Screening.

Rapid and accurate cancer drug screening is of great importance in precision medicine. However, the limited amount of tumor biopsy samples has hindered the application of traditional drug screening methods with microwell plates for individual patients. A microfluidic system provides an ideal platform for handling trace amounts of samples. This emerging platform has a good role in nucleic acid-related and cell related assays. Nevertheless, convenient drug dispensing remains a challenge for clinical on-chip cancer drug screening. Similar sized droplets are merged to add drugs for a desired screened concentration which significantly complicated the on-chip drug dispensing protocols. Here, we introduce a novel digital microfluidic system with a specially structured electrode (a drug dispenser) to dispense drugs by droplet electro-ejection under a high-voltage actuation signal, which can be conveniently adjusted by external electric controls. With this system, the screened drug concentrations span up to four orders of magnitude with small sample consumption. Various amounts of drugs can be delivered to the cell sample with desired amount in a flexible electric control. Moreover, single drug or combinatorial multidrug on-chip screening can be readily achieved. The drug response of normal MCF-10A breast cells and MDA-MB-231 breast tumor cells to two chemotherapeutic substances, cisplatin (Cis) and epirubicin (EP), was tested individually and in combination for proof-of-principle verification. The comparable on-chip and off-chip results confirmed the feasibility of our innovative DMF system for cancer drug screening.

Detection and discrimination of pathogenic bacteria with nanomaterials-based optical biosensors: A review.

Pathogenic bacteria can pose a great threat to food safety and human health. It is therefore imperative to develop a rapid, portable, and sensitive determination and discrimination method for pathogenic bacteria. Over the past few years, various nanomaterials (NMs) have been employed as desirable nanoprobes because they possess extraordinary properties that can be used for optical signal enabled detection and identification of bacteria. By means of modification, NMs can, depending on different mechanisms, sense targets directly or indirectly, which then provides an essential support for the detection and differentiation of pathogenic bacteria. In this review, recent application of NMs-based optical biosensors for food safety bacterial detection and discrimination is performed, mainly in but not limited to noble metal NMs, fluorescent NMs, and point-of-care testing (POCT). This review also focuses on future trends in bacterial detection and discrimination, and machine learning in performing intelligent rapid detection and multiple accurate identification of bacteria.

Sub-5-Minute Ultrafast PCR using Digital Microfluidics.

The development of a rapid and reliable polymerase chain reaction (PCR) method for point-of-care (POC) diagnosis is crucial for the timely identification of pathogens. Microfluidics, which involves the manipulation of small volumes of fluidic samples, has been shown to be an ideal approach for POC analysis. Among the various microfluidic platforms available, digital microfluidics (DMF) offers high degree of configurability in manipulating µL/nL-scale liquid and achieving automation. However, the successful implementation of ultrafast PCR on DMF platforms presents challenges due to inherent system instability. In this study, we developed a robust and ultrafast PCR in 3.7-5 min with a detection sensitivity comparable to conventional PCR. Specifically, the implementation of the pincer heating scheme homogenises the temperature within a drop. The utilization of a µm-scale porous hydrophobic membrane suppresses the formation of bubbles under high temperatures. The design of a groove around the high-temperature zone effectively mitigates the temperature interference. The integration of a soluble sensor into the droplets provides an accurate and instant in-drop temperature sensing. We envision that the fast, robust, sensitive, and automatic DMF system will empower the POC testing for infectious diseases.

Correction: Detection of airborne pathogens with single photon counting and a real-time spectrometer on microfluidics.

Correction for 'Detection of airborne pathogens with single photon counting and a real-time spectrometer on microfluidics' by Ning Yang et al., Lab Chip, 2022, https://doi.org/10.1039/D2LC00934J.

A self-designed device integrated with a Fermat spiral microfluidic chip for ratiometric and automated point-of-care testing of anthrax biomarker in real samples.

A desirable lanthanide-based ratiometric fluorescent probe was designed and integrated into a self-designed Fermat spiral microfluidic chip (FS-MC) for the automated determination of a unique bacterial endospore biomarker, dipicolinic acid (DPA), with high selectivity and sensitivity. Here, a blue emission wavelength at 425 nm was generated in the Fermat spiral structure by mixing the europium (Eu3+) and luminol to form the Eu3+/Luminol sensing probe. DPA in the reservoir can be used to specifically bind to Eu3+ under the negative pressure and transfer energy from DPA to Eu3+ sequentially via an antenna effect, thus resulting in a significant increase in the red fluorescence emission peak at 615 nm. According to the fluorescence intensity ratio (F615/F425), a good linearity can be obtained with increasing the concentration of DPA from 0 to 200 µM with a limit of detection as low as 10.11 nM. Interestingly, the designed FS-MC can achieve rapid detection of DPA in only 1 min, reducing detection time and improving sensitivity. Furthermore, a self-designed device integrated with the FS-MC and a smartphone color picker APP was employed for the rapid automatic point-of-care testing (POCT) of DPA in the field, simplifying complex processes and reducing testing times, thus confirming the great promise of this ready-to-use measurement platform for in situ inspection.

pH Regulator on Digital Microfluidics with Pico-Dosing Technique.

Real-time pH control on-chip is a crucial factor for cell-based experiments in microfluidics, yet difficult to realize. In this paper, we present a flexible pH regulator on a digital microfluidic (DMF) platform. The pico-dosing technology, which can generate and transfer satellite droplets, is presented to deliver alkali/acid into the sample solution to change the pH value of the sample. An image analysis method based on ImageJ is developed to calculate the delivered volume and an on-chip colorimetric method is proposed to determine the pH value of the sample solution containing the acid-base indicator. The calculated pH values show consistency with the measured ones. Our approach makes the real-time pH control of the on-chip biological experiment more easy to control and flexible.

Visual genetic typing of glioma using proximity-anchored in situ spectral coding amplification.

Gliomas are histologically and genetically heterogeneous tumors. However, classical histopathological typing often ignores the high heterogeneity of tumors and thus cannot meet the requirements of precise pathological diagnosis. Here, proximity-anchored in situ spectral coding amplification (ProxISCA) is proposed for multiplexed imaging of RNA mutations, enabling visual typing of brain gliomas with different pathological grades at the single-cell and tissue levels. The ligation-based padlock probe can discriminate one-nucleotide variations, and the design of proximity primers enables the anchoring of amplicons on target RNA, thus improving localization accuracy. The DNA module-based spectral coding strategy can dramatically improve the multiplexing capacity for imaging RNA mutations through one-time labelling, with low cost and simple operation. One-target-one-amplicon amplification confers ProxISCA the ability to quantify RNA mutation copy number with single-molecule resolution. Based on this approach, it is found that gliomas with higher malignant grades express more genes with high correlation at the cellular and tissue levels and show greater cellular heterogeneity. ProxISCA provides a tool for glioma research and precise diagnosis, which can reveal the relationship between cellular heterogeneity and glioma occurrence or development and assist in pathological prognosis.

Fiber Laser-Based Lasso-Shaped Biosensor for High Precision Detection of Cancer Biomarker-CEACAM5 in Serum.

Detection of trace tumor markers in blood/serum is essential for the early screening and prognosis of cancer diseases, which requires high sensitivity and specificity of the assays and biosensors. A variety of label-free optical fiber-based biosensors has been developed and yielded great opportunities for Point-of-Care Testing (POCT) of cancer biomarkers. The fiber biosensor, however, suffers from a compromise between the responsivity and stability of the sensing signal, which would deteriorate the sensing performance. In addition, the sophistication of sensor preparation hinders the reproduction and scale-up fabrication. To address these issues, in this study, a straightforward lasso-shaped fiber laser biosensor was proposed for the specific determination of carcinoembryonic antigen (CEA)-related cell adhesion molecules 5 (CEACAM5) protein in serum. Due to the ultra-narrow linewidth of the laser, a very small variation of lasing signal caused by biomolecular bonding can be clearly distinguished via high-resolution spectral analysis. The limit of detection (LOD) of the proposed biosensor could reach 9.6 ng/mL according to the buffer test. The sensing capability was further validated by a human serum-based cancer diagnosis trial, enabling great potential for clinical use. The high reproduction of fabrication allowed the mass production of the sensor and extended its utility to a broader biosensing field.

Dilute-'N'-Go dideoxy sequencing of all DNA strands generated in multiplex LATE-PCR assays.

We have recently described a Dilute-'N'-Go protocol that greatly simplifies preparation and sequencing of both strands of an amplicon generated using linear-after-the-exponential (LATE)-PCR, an advanced form of asymmetric PCR . The same protocol can also be used to sequence all limiting primer strands in a multiplex LATE-PCR, by adding back each of the depleted limiting primers to a separate aliquot of the multiplex reaction. But, Dilute-'N'-Go sequencing cannot be used directly to sequence each of the excess primer strands in the same multiplex reaction, because all of the excess primers are still present at high concentration. This report demonstrates for the first time that it is possible to sequence each of the excess primer strands using a modified Dilute-'N'-Go protocol in which blockers are added to prevent all but one of the excess primers serving as the sequencing primer in separate aliquots. The optimal melting temperatures, positions and concentrations of blockers relative to their corresponding excess primers are defined in detail. We are using these technologies to measure DNA sequence changes in mitochondrial genomes that accompany aging and exposure to certain drugs.